Sulphur derivatives with a retroamide bond as endothelin-converting enzyme inhibitors

ABSTRACT

Products of formula (1), wherein R 1  is particularly phonyl or biphenyl optionally substituted by halogen n1 and n2 are 0 or 1, R 2  is particularly hydrogen or methyl substituted by phenyl, which may in turn be substituted, and A is partcularly carboxy or alkyl substituted particularly by phenoxy, as well as all isomers and organic and inorganic base addition salts therof, are disclosed.

The present invention relates to new sulphur derivatives containing aretroamide bond, their preparation process, the new intermediatesobtained, their use as medicaments, the pharmaceutical compositionscontaining them and the new use of such derivatives.

A subject of the present invention is the products of formula (I):

in which:

R₁ represents an phenyl or biphenyl radical optionally substituted byone or more radicals chosen from halogen atoms, the following radicals:optionally protected hydroxyl, linear or branched alkoxy containing upto 4 carbon atoms, cyano, free, salified, esterified or amidifiedcarboxy, benzyloxy and the dioxol radical, n1 and n2, identical ordifferent, represent the integer 0 or 1,

R₂ represents a hydrogen atom or a methyl radical substituted by aphenyl, phenylthio or indolyl radical and optionally by a second phenylradical, these phenyl, phenylthio and indolyl radicals being optionallysubstituted by one or more radicals chosen from halogen atoms and thefollowing radicals: optionally protected hydroxyl, linear or branchedalkoxy containing up to 4 carbon atoms, cyano, free, salified,esterified or amidified carboxy, benzyloxy, thienyl, naphthyl andphenyl, these three last radicals being themselves optionallysubstituted by one or more radicals chosen from halogen atoms and thefollowing radicals: optionally protected hydroxyl, linear or branchedalkoxy containing up to 4 carbon atoms, cyano and free, salified,esterified or amidified carboxy,

A represents the free, salified, esterified or amidified carboxyradical, the free or salified tetrazolyl radical, or an alkyl radical,containing up to 10 carbon atoms and substituted by a radical chosenfrom the following radicals: free, salified, esterified or amidifiedcarboxy, the optionally protected hydroxyl, alkoxy containing up to 4carbon atoms, phenoxy, phenyl, naphthyl, thienyl, indolyl and pyridyl,these radicals being optionally substituted by one or more radicalschosen from halogen atoms and the following radicals: optionallyprotected hydroxyl, linear or branched alkoxy containing up to 4 carbonatoms, cyano and free, salified, esterified or amidified carboxy,

1 and 2 indicating, if appropriate, the asymmetric centres of theproducts of formula (I), said products of formula (I) being in allpossible racemic, enantiomeric and diastereoisomeric isomer forms, aswell as the addition salts with mineral and organic acids or with themineral and organic bases of said products of formula (1).

In the products of formula (I) and in what follows:

the term linear or branched alkyl radical designates the followingradicals: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, hexyl, isohexyl and also heptyl, octyl,nonyl and decyl as well as their linear or branched position isomers,

the term linear or branched alkoxy radical designates the followingradicals: methoxy, ethoxy, propoxy, isopropoxy, linear, secondary ortertiary butoxy, pentoxy or hexoxy as well as their linear or branchedposition isomers,

the term halogen atom preferably designates the chlorine atom, but canalso represent a fluorine, bromine or iodine atom.

The hydroxyl radical can in particular be in the form of thetrifluoromethylsulphonyloxy radical.

The carboxy radical or radicals of the products of formula (I) can besalified or esterified by the various groups known to a person skilledin the art among which there can be mentioned, for example:

among the salification compounds, mineral bases such as, for example, anequivalent of sodium, of potassium, of lithium, of calcium, of magnesiumor of ammonium or organic bases such as, for example, methylamine,propylamine, trimethylamine, diethylamine, triethylamine,N,N-dimethylethanolamine, tris (hydroxymethyl) amino methane,ethanolamine, pyridine, picoline, dicyclohexylamine, morpholine,benzylamine, procaine, lysine, arginine, histidine, N-methyl-glucamine.

The sodium or potassium salts are preferred,

among the esterification compounds, the alkyl radical in order to formalkoxy carbonyl or arylalkoxycarbonyl groups, such as, for example,methoxycarbonyl, ethoxycarbonyl, n-propoxy- and isopropoxy-carbonyl,n-butoxy-, isobutoxy- and tert-butoxy-carbonyl or benzyloxycarbonyl,these alkyl radicals can be substituted by radicals chosen for examplefrom halogen atoms, hydroxyl, alkoxy, acyl, acyloxy, alkylthio, amino oraryl radicals such as, for example, in the chloromethyl, hydroxypropyl,methoxymethyl, propionyloxymethyl, methylthiomethyl, dimethylaminoethyl,benzyl or phenethyl groups.

There can also be mentioned the radicals formed with the remainders ofeasily cleavable esters such as the methoxymethyl, ethoxymethylradicals; the acyloxyalkyl radicals such as pivaloyloxymethyl,pivaloyloxyethyl, acetoxymethyl or acetoxyethyl; the alkyloxycarbonyloxyalkyl radicals such as methoxycarbonyloxy methyl or ethyl radicals, theisopropyloxycarbonyloxy methyl or ethyl radicals.

A list of such ester radicals can be found for example in the EuropeanPatent EP 0 034 536.

By amidified carboxy is meant the groups of —CON(R₆) (R₇) type in whichthe identical or different R₆ and R₇ radicals represent a hydrogen atomor an alkyl radical having from 1 to 4 carbon atoms such as the methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butylradicals.

Among the —CON(R₆) (R₇) groups defined above, those in which the —N(R₆)(R₇) radical represents the amino, mono or dimethylamino radical arepreferred.

The N(R₆) (R₇) radical can also represent a heterocycle which may or maynot contain an additional heteroatom. There can be mentioned thepyrrolyl, imidazolyl, indolyl, piperidino, morpholino, piperazinylradicals. The piperidino or morpholino radicals are preferred.

Examples of the protective group of the protected hydroxyl radical aregiven in particular in the usual book known to a person skilled in theart: Protective Groups in Organic Synthesis, Theodora W. Greene, HarvardUniversity, printed in 1981 by Wiley-Interscience Publishers, John Wiley& Sons.

The addition salts with mineral or organic acids of the products offormula (I) can be, for example, the salts formed with the followingacids: hydrochloric, hydrobromic, hydroiodic, nitric, sulphuric,phosphoric, propionic, acetic, formic, benzoic, maleic, fumaric,succinic, tartaric, citric, oxalic, glyoxylic, aspartic, ascorbic,alkylmonosulphonic acids such as for example methanesulphonic acid,ethanesulphonic acid, propanesulphonic acid, alkyldisulphonic acids suchas for example methanedisulphonic acid, alpha, beta-ethanedisulphonicacid, arylmonosulphonic acids such as benzenesulphonic acid andaryldisulphonic acids.

More particularly there can be mentioned the salts formed withhydrochloric or methanesulphonic acids for example.

It should be remembered that the stereoisomerism can be defined in itswidest sense as the isomerism of compounds having the same structuralformulae, but the different groups of which are arranged differently inspace.

It is understood that the definition of the products of formula (I) asdefined above includes all possible stereoisomers, all racemicmodifications, all optical isomers and all mixtures of these productswhich would have the activity indicated hereafter.

The products of formula (I) contain in particular two centres {circlearound (1)} and {circle around (2)}, {circle around (1)} beingasymmetrical and {circle around (2)} being asymmetrical when R₂ does notrepresent a hydrogen atom. A particular subject of the present inventionis the products of formula (I) in which the first asymmetrical centre{circle around (1)} is preferably in R form, the second centre {circlearound (2)} can preferably be in racemic or enantiomeric R or S form.

A particular subject of the present invention is the products of formula(I) as defined above, in which R₁, R₂, and n2 have the meaningsindicated above, n1 represents the integer 1, and A represent the free,salified, esterified ou amidified carboxy radical or an alkyl radical,containing at most 10 carbon atoms and substituted by a radical chosenfrom the free, salified, esterified ou amidified carboxy radicals,optionally protected hydroxyl radicals, alkoxy radicals containing atmost 4 carbon atoms, and the phenoxy radical, said products of formula(I) being in all possible racemic, enantiomeric and diastereoisomericisomer forms, as well as the addition salts with mineral and organicacids or with mineral and organic bases of said products of formula (I).

A more particular subject of the present invention is the products deformula (I) as defined above, in which R₁ represents a phenyl orbiphenyl radical, optionally substituted by a halogen atom, or by ahydroxyl radical optionally in the form of thetrifluoromethyl-sulphonyloxy radical,

n1 represents the integer 1,

n2 represents the integer 0,

R₂ represents a hydrogen atom or a methyl radical substituted by aphenyl radical, itself optionally substituted by a thienyl or phenylradical itself optionally substituted by a cyano radical,

A represents the free, salified, esterified ou amidified carboxy radicalor an alkyl radical, containing at most 10 carbon atoms substituted by aphenoxy radical,

said products of formula (I) being in all possible racemic, enantiomericand diastereoisomeric isomer forms, as well as the addition salts withmineral and organic bases of said products of formula (I).

In particular, R₁ can represent a phenyl or biphenyl radical, optionallysubstituted by a bromine atom.

A quite particular subject of the present invention is the products deformula (I) as defined above, the names of which follow:

[S-(R*,S*)]N-[1-(mercaptomethyl)-2-phenylethyl]-alpha-[[(phenylmethoxy)carbonyl]amino]-1H-indole-3-propanamide,

2′-cyano-alpha-[[[1-(mercaptomethyl)-2-phenylethyl]amino]carbonyl](1,1′-biphenyl)-4-propanoicacid,

(R)N-(1-mercaptomethyl)-2-phenylethyl)-11-phenoxy-undecanamide,

said products of formula (I) being in all possible racemic, enantiomericand diastereoisomeric isomer forms, as well as the addition salts withmineral and organic bases of said products of formula (I).

A subject of the present invention is also the preparation process forthe products of formula (I), such as defined above, characterized inthat a product of formula (II)

in which n1 has the meaning indicated above and R′₁ has the meaningindicated above for R₁ in which the optional reactive functions areoptionally protected, is subjected either to the action of a compound offormula (III):

in which R′₂ has the meaning indicated above for R₂ in which theoptional reactive functions are optionally protected, and R representsan alkyl or arylalkyl radical, in order to obtain the product of formula(IV):

in which n1, R′₁, R′₂ and R have the meanings indicated above, or to theaction of an acid halide of formula (V):

in which n2 has the meaning indicated above, Hal represents a halogenatom, R′₂ has the meaning indicated above and A′ has the meaningindicated above for A in which the optional reactive functions areoptionally protected, such as in particular A′ does not represent a freecarboxy radical, in order to obtain a product of formula (VI):

in which n1, n2, R′₁, R′₂ and A′ have the meanings indicated above,

which products of formula (IV) and (VI), in order to obtain products offormula (I) or to convert the products of formula (I) into otherproducts of formula (I), can be treated, if desired and if necessary, toone or more of the following reactions, in any order:

a saponification reaction of the ester function into an acid function,

a conversion reaction of the cyano function or amide function into anacid or tetrazolyl function,

a conversion reaction of the alkoxy function into the hydroxyl function,

an esterification, salification or amidification reaction of the acidfunction,

a reaction which releases the thiol function from the

an elimination reaction of the protective groups which can be carried bythe protected reactive functions,

a salification reaction by a mineral or organic acid or base in order toobtain the corresponding salt, said products of formula (I) thusobtained being in all possible racemic, enantiomeric anddiastereoisomeric isomer forms.

Under the preferred conditions for implementing the invention, theprocess described above can be carried out in the following fashion:

the reaction of the product of formula (I) as defined above with theproduct of formula (III) as defined above is preferably carried out witha coupling agent such as for example EDC in methylene chloride or alsoBOP methyl cyanide in the presence of triethylamine, or also DDC,

the reaction of the product of formula (II) as defined above with theproduct of formula (V) as defined above is preferably carried out, inmethylene chloride in the presence of pyridine. In the compound offormula (V), the halogen atom is preferably a chlorine atom.

According to the values of R′₁, R′₂ and A′, the products of formulae(IV) and (VI) constitute or do not constitute products of formula (I)and can produce products of formula (I), or be converted into otherproducts of formula (I) by being subjected to one or more of thereactions indicated above which can be carried out, for example, asindicated hereafter.

The various reactive functions which can be carried by certain compoundsof the reactions defined above can, if necessary, be protected: it isfor example the hydroxyl or free carboxy radicals which can be protectedby the appropriate protective groups.

The following non-exhaustive list, of examples of the protection ofreactive functions can be mentioned:

the amine groups can be protected in the form of other carbamates, suchas those known in the chemistry of peptides,

the hydroxyl groups can be protected for example by alkyl radicals suchas tert-butyl, trimethylsilyl, tert-butyldimethylsilyl, methoxymethyl,tetrahydropyrannyl, benzyl or acetyl.

The products described above can, if desired, be the object, on theoptional carboxy functions, of esterification, salification oramidification reactions, which can be carried out according the usualmethods known to a person skilled in the art.

The acid functions of the products described above can be, if desired,amidified by a primary or secondary amine for example in methylenechloride in the presence of, for example,1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride at ambienttemperature.

The acid functions acid can be protected for example in the form ofesters formed with easily cleavable esters such as benzylic or terbutylic esters or esters known in the chemistry of the peptides.

The optional saponification reactions of the ester function into an acidfunction of the products described above can, if desired, be carried outunder the usual conditions known to a person skilled in the art inparticular by acid or alkaline hydrolysis for example with soda orpotash in alcoholic medium such as, for example, in methanol or alsowith hydrochloric or sulphuric acid.

The optional cyano functions of the products described above can, ifdesired, be converted into an acid function under the usual conditionsknown to a person skilled in the art for example by a double hydrolysiscarried out in acid medium such as for example in a sulphuric acid,glacial acetic acid and water mixture, these three compounds preferablybeing in equal proportions, or also in a soda, ethanol and water mixtureunder reflux.

The optional cyano functions of the products described above can, ifdesired, be converted into the tetrazolyl function under the usualconditions known to a person skilled in the art such as for example bythe cycloaddition of a metal azide such as for example sodium azide ortrialkyltin azide on the nitrile function as indicated in the methoddescribed in the article referenced as follows: J. OrganometallicChemistry., 33, 337 (1971) KOZIMA S.and al.

The acid functions can be converted into tetrazole as indicated in thepublication Bio. Med. Chem. Leh. 1995, 145.

The optional alkoxy functions such as in particular methoxy of theproducts described above can, if desired, be converted into the hydroxylfunction under the usual conditions known to a person skilled in the artfor example with boron tribromide in a solvent such as for examplemethylene chloride, with pyridine hydrobromide or hydrochloride or alsowith hydrobromic or hydrochloric acid in water or trifluoroacetic acidunder reflux.

The optional hydroxyl functions of the products described above can, ifdesired, be converted into an acid function by oxidation under the usualconditions known to a person skilled in the art in the conditions suchas for example by the action of Jones reagent to access the acids.

The elimination of the protective groups such as for example thoseindicated above can be carried out under the usual conditions known to aperson skilled in the art in particular by an acid hydrolysis carriedout with an acid such as hydrochloric, benzene sulphonic or paratoluenesulphonic, formic or trifluoroacetic acid or also by catalytichydrogenation.

The phthalimido group can be eliminated with hydrazine.

A list of the different protective groups that can be used will be foundfor example in the Patent BF 2 499 995.

The products described above can, if desired, be the object ofsalification reactions for example with a mineral or organic acid orwith a mineral or organic base according to the usual methods known to aperson skilled in the art.

The optional optically active forms of the products described above canbe prepared by resolution of the racemics according to the usual methodsknown to a person skilled in the art.

Illustrations of such reactions defined above are given in thepreparation of the examples described hereafter.

The structures and properties of endothelin and of its precursor BigEndothelin are described in the literature such as for example in thedocument WO 93/11154.

The products of formula (I) of the present invention have been found tohave an inhibitory activity on the enothelin-converting enzyme whichallows the properly so-called endothelin to be obtained from BigEndothelin, which is thus an extremely powerful vasoconstrictor agent.

The products of formula (I) of the present invention can therefore beused in the treatment of illnesses resulting from abnormally highquantities of endothelin.

The compounds of formula (I) as defined above as well as their additionsalts as defined above have useful pharmacological properties.

The products of formula (I) as defined above, endowed with inhibitoryproperties of the endothelin-converting enzyme, can thus in particularreduce the quantities and therefore the effects of endothelin, inparticular the vasoconstrictor and hypertensor effects induced byendothelin. In particular an antiischemic effect is noted.

The products of formula (I) therefore also have the effect of reducingthe stimulating effects of endothelin at the level of all cell types, inparticular the smooth muscle cells, the fibroblasts, the neuronal cellsand the bone cells.

These properties justify their use in therapeutics and a particularsubject of the invention is as medicaments, the products of formula (I),

said products of formula (I) being in all possible racemic, enantiomericand diastereoisomeric isomer forms, as well as the addition salts withpharmaceutically acceptable mineral and organic acids or withpharmaceutically acceptable mineral and organic bases of said productsof formula (I).

More particularly a subject of the invention is also, as medicaments,the preferred products as defined by formula (I) above in which;

R₁ represents a phenyl or biphenyl radical, optionally substituted byhalogen atom,

n1 represents the integer 1,

n2 represents the integer 0,

R₂ represents a hydrogen atom or a methyl radical substituted by aphenyl radical, itself optionally substituted by a thienyl or phenylradical itself optionally substituted by a cyano radical,

A represents the free, salified, esterified or amidified carboxy radicalor an alkyl radical, containing at most 10 carbon atoms substituted by aphenoxy radical,

said products of formula (I) being in all possible racemic, or opticallyactive isomer forms, as well as the addition salts with pharmaceuticallyacceptable mineral and organic acids or with pharmaceutically acceptablemineral and organic bases of said products of formula (I).

A quite particular subject of the invention is, as medicaments, theproducts described hereafter in the examples and in particular theproducts of formula (I) as defined above, the names of which follow:

[S-(R*,S*)]N-[1-(mercaptomethyl)-2-phenylethyl]-alpha-[[(phenylmethoxy)carbonyl]amino]-1H-indole-3-propanamide,

2′-cyano-alpha-[[[1-(mercaptomethyl)-2-phenylethyl]amino]carbonyl](1,1′-biphenyl)-4-propanoicacid,

(R)N-(1-mercaptomethyl)-2-phenylethyl)-11-phenoxy-undecanamide,

as well as the addition salts with pharmaceutically acceptable mineraland organic bases of said products of formula (I).

The medicaments, which are a subject of the invention, therefore findtheir use in the treatment, by use of an inhibitory agent of theendothelin-converting enzyme, for illnesses such as, for example,vascular spasms, vasospasm as a result of a cerebral haemorrhage,coronary spasms, peripheral vascular spasms as well as renalinsufficiencies. These medicaments can also be used in the treatment ofmyocardial infarction, of congestive cardiac insufficiency, in theprevention of post-angioplasty recurrence of stenosis, of cardiac andvascular fibrosis, in the treatment of atherosclerosis, of certain formsof hypertension such as in particular pulmonary hypertension, as well asin the treatment of asthma.

The medicaments, which are a subject of the invention, can also find ause in the treatment of osteoporosis, prostatic hypertrophia and asneuronal protectors.

The invention extends to the pharmaceutical compositions containing asactive ingredient at least one of the medicaments as defined above.

These pharmaceutical compositions can be administered by buccal, rectalroute, by parenteral route or by local route as a topical application onthe skin and mucous membranes or by injection by intravenous orintramuscular route.

These compositions can be solid or liquid and be presented in all thepharmaceutical forms commonly used in human medicine, such as forexample, plain or sugar-coated tablets, capsules, granules,suppositories, injectable preparations, ointments, creams, gels andaerosol preparations; they are prepared according to the usual methods.The active ingredient can be incorporated with excipients usuallyemployed in these pharmaceutical compositions, such as talc, gum arabic,lactose, starch, magnesium stearate, cocoa butter, aqueous ornon-aqueous vehicles, fatty substances of animal or vegetable origin,paraffin derivatives, glycols, various wetting, dispersing oremulsifying agents, preservatives.

The usual dose, variable according the product used, the patient treatedand the illness in question, can be, for example, from 1 to 300 mg perday in an adult, by oral route or from 1 to 100 mg per day byintravenous route.

Certain starting products of formula (II), (III) and (V) are known, maybe commercially available or prepared according to the usual methodsknown to a person skilled in the art or also for example as indicated inthe European Patent EP 0465369.

In particular certain products of formula (II) can also be prepared fromother products of formula (II) for example by subjecting them to one ormore of the reactions described above, carried out under the conditionsalso described above.

Preparations for the compounds of formula (II) are indicated in theexamples described hereafter.

It should be noted that the compound of formula (II) can be in racemicor optically pure form and that, consequently, the product of formula(IV) obtained can also be in racemic or optically pure form.

The compound of formula (II) in which ni represents the integer 1 andR′₁ represents the phenyl radical can be prepared as indicated inPreparation 1E of EP 0465369, either starting with the amino acidL-phenylalamine in order to produce the product of formula (II) of formS, or starting with the amino acid D-phenylalamine in order to producethe product of formula (II) of form R, or starting with a mixture ofamino acids, L- and D-phenylalamine in order to produce the product offormula (II) of racemic form: these compounds of formula (II) thusobtained produce respectively, after reaction with the compound offormula (III), a product of formula (IV) which will be respectively in{circle around (1)} in S form (Example 1), in R form (Example 2) or inracemic form (Example 5).

Other compounds of formula (II), can be obtained in the same fashionstarting from other amino acids.

In particular, the products of formula (II) in which R′₁ represents thebiphenyl radical can be obtained starting from tyrosine.

Such a reaction is indicated in the preparation of Example 7, describedhereafter.

Other compounds of formula (II), in which n1 represents the integer 0can be prepared starting from the corresponding acid in order to produceby a Curtius reaction described in the literature, the sought amine.

The compounds of formula (III) represent amino acids which can besubstituted on the nitrogen: such compounds of formula (III) may becommercially available or be prepared according to the usual methodsknown to a person skilled in the art.

The compounds of formula (V) are preferably acid chlorides prepared fromthe corresponding acid itself commercially available or preparedaccording to the usual methods known to a person skilled in the art,such as by condensation of an alkyl halide on a malonate.

Finally, a subject of the present invention is, as new industrialproducts, the compounds of formulae (IV) and (VI) as defined above.

Particularly a subject of the present invention is the use of theproducts of formula (I) as defined above, for the preparation of aninhibitory agent of the endothelin-converting enzyme.

Therefore a particular subject of the present invention is the use ofthe products of formula (I) as defined above, for the preparationpharmaceutical compositions intended for the treatment, by inhibition ofthe endothelin-converting enzyme, for illnesses such as, in particular,hypertension induced by endothelin, vascular spasms, the effects of acerebral haemorrhage, renal insufficiencies, myocardial infarction ,cardiac insufficiency as well as the prevention of post-angioplastyrecurrence of stenosis and cardiac and vascular fibrosis.

The following examples illustrate the invention without however limitingit.

EXAMPLE 1 [S-N-(1-Mercaptomethyl)-2-phenylethyl)-2-(((phenylmethoxy)carbonyl)amino)-indole-3-propanamide

STAGE 1

[-(R*,R*)]N-[1-[(Acetylthio)methyl)-2-phenyl-ethyl]-alpha-[[(phenylmethoxy)carbonyl]amino]-1H-indole-3-propanamide

183 mg of 1-(3-dimethylamino propyl)3-ethyl carbodiimide hydrochlorideis introduced into 10 ml of methylene chloride and 120 μl oftriethylamine is added. Agitation is carried out for 10 minutes atambient temperature and 300 mg of N-carbobenzyloxy Triptomane (NCBz Trp)is added. The reaction medium is again left for 10 minutes at ambienttemperature then in one go 200 mg of the amine hydrochloride in S formis added, prepared as indicated in Preparation 1E of EP 0465369 withL-phenylalamine as starting product. The reaction is agitated for 4hours at ambient temperature, followed by evaporating, taking up in 30m1 of ethyl acetate and washing twice with 0.1N hydrochloric acid, thenwith salt water (twice). After chromatography on silica with ethylacetate-hexane: 50—50 as eluant, 270 mg of expected product is obtained.

STAGE 2

[S-N-(1-Mercaptomethyl)-2-phenylethyl)-2-(((phenyl-methoxy)Carbonyl)amino)-indole-3-propanamide

120 mg of product obtained in Stage 1 above is introduced into 3 ml ofmethanol. Then 0.34 ml of 1N soda is added and the reaction medium isleft for 1 hour at 0° C. After taking up in 4 ml of water, acidificationis carried out with 1N hydrochloric acid until a pH approx.=½ and theprecipitate formed is filtered off. After drying, 120 mg of expectedproduct is obtained. M.p.=164° C. Rotatory power α_(D) at aconcentration of 0.5 in methanol=−20.8°.

ANALYSES IR (CHCl₃) cm−¹

═C—NH 3477-3420 >═O 1716-1673 SH 2570 Aromatic C═C 1620-1578-1585-1497

Amide II

NMR (250 MHz 1H, CDCl₃) 0.81 (t, 1H,SH); 2.09 to 2.37 (m, CHCH₂—SH)−2.64 (d, 2H, CH₂); 3.11 (dd, 1H,CH₂); 3.32 (dd, 1H); 4.20 (m 1H); 4.45(n, 1H); 5.12 (s, 2H, O—CH₂-ph); 5.43 (d, 1H,NH); 5.75 (d, 1H,NH); 6.96to 7.66 (n, 14H); 8.07 (ws, 1H, indole NH).

EXAMPLE 2[(R*,S*)]-N-[1-(Mercaptomethyl)-2-phenylethyl]-alpha-[[(phenylmethoxy)carbonyl)amino)-1H-indole-3-propanamide

The operation is carried out as in Example 1 using in Stage 1 the aminehydrochloride in R form prepared as indicated in Preparation 1E of EP0465369 with as starting product D-phenylalamine. In this way 120 mg ofexpected product is obtained.

Rotatory power α_(D) at a concentration of 0.75 in chloroform=+8.50°.

ANALYSES

NMR (1H, CDCl₃, 250 MHz, δ ppm) 1.04 (t, 1H,SH); 2.3 to 2.7 (m, 4H,2×CH₂); 3.11 (dd, 1H); 3.34 (dd, 1H); 4.18 (m, 1H,CH—CH₂); 4.44 (m,1H,CH—NHCHO); 5.11 (s, 2H, CH₂ ph); 5.40 (d, 1H,NH); 5.75 (d, 1H,NH);8.02 (s, 1H, indole NH); 6.95 (n, 3H, aromatic H); 7.10 to 7.4 (n, 11H,aromatics H's); 7.66 (wd, 1H, aromatics).

EXAMPLE 3 [S-(R*,R*)]-2-(((1,1-Dimethylethoxy)carbonyl)amino)-N-(l-(mercaptomethyl)-2-phenylethyl)-indole-3-propanamide

STAGE 1

[S-(R*,R*)]-N-[1-((Acetylthio)methyl]-2-phenylethyl]-alpha-[[(1,1-dimethylethoxy)carbonyl)amino]-1H-indole-3-propanamide

The operation is carried out as in Stage 1 of Example 1 usingN-BOC-tryptophan (N-terbutyloxycarbonyl-Trp) instead of N-CBZ-tryptophan(N-carbobenzyloxy-Trp). In this way 270 mg of expected product isobtained.

STAGE 2

[S-(R*,R*)]]-2-(((1,1-Dimethylethoxy)carbonyl)amino)-N-(1-(mercaptomethyl)-2-phenylethyl)-indole-3-propanamide

The operation is carried out as in Stage 2 of Example 1 and in this way120 mg of expected product is obtained. M.p.=77° C.

Rotatory power α_(D) at a concentration of 0.5 in methanol=−22.4°

ANALYSES

NMR (1H, CDCl₃, 250 MHz, δ ppm) 0.87 (t, 1H,SH); 1.44 (s, 9H, tbu); 2.26(m, CH₂,2H); 2.65 (m, 2H,CH₂-ph); 3.10 (dd, 1H,CH₂); 3.30 (dd, 1H,CH₂);4.22 (m, 1, 1H,CH); 4.39 (m, CH,LH); 5.13 (d, 1H,NH); 5.84 (d, 1H,NH);6.99 (s, 1H,H₂ indole); 7 to 7.22 (m, 7H); 7.36 (d, 1H,Ar); 7.67 (s,1H,Ar); 8.04 (ws, 1H,NH indole). IR (CHCl₃) cm⁻¹

═C—NH 3477-3420 >═O 1706-1672 SH 2570 Conjugated syst. Amide II +1620-1605-1513-1496-1490 Aromatic Me of tBu 1368

EXAMPLE 42′-cyano-alpha-[[[1-(mercaptomethyl)-2-phenylethyl]amino]carbonyl]-(1,1′-biphenyl)-4-propanoicacid

STAGE 1

2′-cyano-alpha-(ethoxycarbonyl)-(1,1′-biphenyl)-4-propanoyl chloride

105 mg of sodium hydride at 60% in oil, washed twice beforehand withpentane, is introduced into 15 ml of tetrahydrofuran and placed at 0° C.under argon. A solution of 0.5 ml of terbutyl ethyl malonate is addeddropwise to 1 ml of tetrahydrofuran and the whole is left for 30 minutesat ambient temperature then returned to 0° C. and a solution of 410 mgof 4′-(bromomethyl)-(1,1′-biphenyl)-2-carbonitrile prepared as indicatedin EP 0465368 (Example 49) is added dropwise. The solution is agitatedfor 1 hour at 0° C. and for 3 hours at ambient temperature, thenacidified with 1N hydrochloric acid to a pH˜2.Then extraction is carriedout with ethyl acetate followed by drying. After chromatography onsilica with ethyl acetate—cyclohexane: 25-75 as eluant, 400 mg of an oilis obtained which is taken up in 10 ml of methylene chloride. 10 ml oftrifluoroacetic acid is added and the whole is left for 3 hours atambient temperature then evaporation is carried out. The oily residue istaken up in 10 ml of thionyl chloride and left under agitationovernight. After evaporation of the thionyl chloride, the expected acidchloride is obtained used as it is in the following stage.

STAGE 2

Ethyl [R-(R*,R*)] and (R-(R*,S*)]alpha-[[[1-((acetylthio)methyl]-2-phenylethyl]amino]carbonyl)-2′-cyano-(1,1′-biphenyl)-4-propanoate

10 ml of methylene chloride is added to the product obtained in Stage 1above and, after having taken the reaction medium to reflux, 377 mg ofthe amine hydrochloride obtained in Preparation 1E of EP 0465369 isadded. Then a few drops of pyridine is added in order to obtain a pH˜4-6and agitation is carried out under reflux for 4 hours. The solution isthen evaporated and after chromatography on silica with ethylacetate—cyclohexane—methylene chloride: 25-25-50 as eluant, 400 mg ofexpected product is obtained.

Analyses

NMR (1H, CDCL₃, 200 MHz) 1.2 (2t, 3H, CH₃); 2.4 and 2.45 (2s, 3H, SAc);2.7-3.5 (m, 7H); 4.2 (m, 2H, CO₂CH₂); 4.4 (m, 1H, OC—CH—CO); 6.55 and6.65 (2d, 1H, NH); 7.1 to 7.6 (m, 11H); 7.7 (d, 1H, Ar); 7.9 (d, 1H,Ar).

STAGE 3

2′-Cyano-alpha-[[[1-(mercaptomethyl)-2-phenylethyl]amino]carbonyl]-(1,1′-biphenyl)-4-propanoic Acid

400 mg of product obtained in Stage 2 above is introduced into 5 ml oftetrahydrofuran and 2.5 ml of water is added. Then 115 mg of lithiumhydroxide is added at ambient temperature and the whole is left underagitation for 2 hours.

The reaction is then acidified by the addition of 1N hydrochloric acidto pH˜2,then, after the addition of 5 ml of H₂O, extraction is carriedout three times with methylene chloride+10% of methanol.

After drying and evaporation, chromatography is carried out on silicawith methylene chloride—methanol: 90-10 as eluant and 280 mg of expectedproduct (white foam) is obtained in the form of a mixture of 2diastereoisomers. Tgum˜130° C.

Analyses:

NMR (DMSO, 300 MHz, δ ppm) 1.98 (m, SH); 2.4 to 3.3 (m, 7H); 3.93 and4.02 (m, 1H, OC—CH—CO); 7.08 to 7.32 (m, 7H, Ar); 7.41 (m, 2H, Ar); 7.55(m, 2H, Ar); 7.77 (m, 1H); 7.92 (d, 1H, Ar); 8.32 and 8.38 (wd, 1H,CONH).

EXAMPLE 5 (S) Alpha-(((1-(mercaptomethyl)-2-phenylethyl) Amino)Carbonyl) Benzenepropanoic Acid

STAGE 1

Alpha-(ethoxycarbonyl)-benzenepropanoyl Chloride

The operation is carried out as in Stage 1 of Example 4 using benzylbromide (commercial) instead of 4′-(bromomethyl) (1,1′-biphenyl)2-carbonitrile and in this way the expected product is obtained.

STAGE 2

Ethyl [S-(R*,S*)] and (S-(R*,S*)]alpha-[[[1-[(acetylthio)Methyl]-2-phenylethyl] Amino]carbonyl]-benzenepropanoate

The operation is carried out as in Stage 2 of Example 4 and in this waythe expected product is obtained.

STAGE 3

(S) Alpha-(((1-(mercaptomethyl)-2-phenylethyl)amino)carbonyl)benzenepropanoic Acid

The operation is carried out as in Stage 3 of Example 4 and in this waythe expected product is obtained. M.p.=65-70° C.;

Analyses:

NMR (DMSO, 300 MHz, δ ppm) Mixture of 2 diastereoisomers (S,R) and (S,S)1.75 and 2.22 (wt, SH); 2.3 to 4.5 (m, 8H); 7 to 7.30 (m, 10H); 8.09 and8.56 (d, 1H, CONH); 12.52 (wide m, COOH).

EXAMPLE 6 (R)N-(1-Mercaptomethyl)-2-phenylethyl)-11-phenoxy-undecanamide

STAGE 1

11-phenoxy-undecanoyl Chloride

250 mg of 11-phenoxy undecanoic acid (commercial) is mixed with 2 ml ofpure thionyl chloride and the whole is left overnight at ambienttemperature. In this way 265 mg of expected product is obtained.

STAGE 2

(R) N-[1-[(Acetylthio) methyl]-2-phenylethyl]-11-phenoxy-undecanamide

The operation is carried out as in Stage 2 of Example 4 starting with265 mg of the product obtained in Stage 1 above and 120 mg of the aminehydrochloride (R form) obtained in Preparation 1E of EP 0465369.In thisway 150 mg of expected product is obtained.

STAGE 3

N-(1-Mercaptomethyl)-2-phenylethyl) 11-phenoxy-undecanamide

100 mg of the product obtained in Stage 2 above is introduced into 4 mlof methanol and 120 μl of 2N soda is added at 0° C. The whole isagitated for 1 hour at 0° C. Then 10 ml of water is added, the whiteprecipitate which forms is filtered out and dried. In this way 70 mg ofexpected product is obtained. M.p.=65° C.

NMR (CDCl₃, 300 MHz, δ ppm); 1.20 to 1.82 (m, 17H); 2.14 (t, COCH₂);2.66 (m, AB/sys resolved, CH,CH ₂SH); 2.88 (m, ph-CH ₂,CH); 3.95 (t,CH₂-CH₂-OPh); 4.37 (m, CH₂-CH(NH)CH₂); 5.55 (d, NHCO); 6.85 to 6.97 (m,2H); 7.16 to 7.35 (m, 8H).

EXAMPLE 7 2′-cyano-alpha-[[[1-(mercaptomethyl)2-biphenylethyl]amino]carbonyl]-(1,1′-biphenyl)-4-propanoic Acid

The operation is carried out as in Example 4,taking as starting productthe amine hydrochloride in which R₁ represent biphenyl, instead of theamine hydrochloride in which R₁ represent phenyl, prepared by using asstarting product the product(R)-α-[[(1,1-dimethylethoxy)carbonyl]amino)(1,1′-biphenyl ]-4-propanoicacid, which here is in the R form and which is prepared as itsenantiomer of S form the preparation of whch is described in thefollowing reference: Journal of Medecinal Chemistry, 1995, Vol. 38,1689,then by preparing the corresponding hydrochloride as indicated inStage D of EP 0465369. In this way the expected product of Example 7 isobtained.

By proceeding as in Examples 4 and 7 described above, using, ifappropriate, instead of 4′-(bromo-methyl) (1,1′-biphenyl)2-carbonitrile, 4′-2-thienyl benzyl bromide, prepared according to theprocess described in the following reference: Journal of MedicinalChemistry 38, 2357, 1995, using the 2-thienyl boronic as arylboronic, inthis way the following products of Examples 8, 9 and 10 are obtained:

EXAMPLE 8 [[(1-(Mercaptomethyl)-2-biphenylethyl]amino]carbonyl]-(1,1′-thienylphenyl)-4-propanoic acid

Analyses:

MS: (M-CO₂)H⁺:458

EXAMPLE 9 ([[[1- (Mercaptomethyl) 2-phenylethyl]amino]carbonyl](1,1-thienylphenyl) 4-propanoic acid EXAMPLE 102′-Cyano-alpha-[[[1-(mercaptomethyl) 2-(paratrifluoromethylsulphonyloxy)phenylethyl]amino]carbonyl](1,1′-biphenyl) 4-propanoic acid

Analyses:

NMR (DMSO) 2.6 to 3.2 (7H); 3.92 and 4.04 (m, 1H); 7.06 to 7.95 (Ar)8.25 and 8.4 (1H,NH).

EXAMPLE 11 OF PHARMACEUTICAL COMPOSITION

Tablets corresponding to the following formula were prepared:

Product of Example 4  50 mg Excipient for a tablet completed at 200 mg

(detail of excipient: lactose, talc, starch, magnesium stearate).

PHARMACOLOGICAL RESULTS Determination of the Inhibitory Effect of theEndothelin-converting Enzyme (ECE)

A test is used, in which the product (2,3-³H)propionyl-b-ET-1(19-35)prepared as indicated below in a) is cleaved by ECE into the product(2,3-³H)propionyl-b-ET-1(19-21) in the presence of product P of whichthe inhibitory activity of ECE one wishes to determine as indicatedbelow in b): the inhibitory activity of ECE of the product P willtherefore be accordingly as high as the quantity of product of formula(II) formed, determined by counting the radioactivity, will be low. Theoperation is carried out as follows:

a)—Preparation of Tritiated Peptide (2,3-³H)Propionyl-b-ET-1 (19-35)

N-succinimidyl- (2, 3-³H)-propionate (Amersham code TRK.556) is insolution in 5 ml of toluene at 1 mCi/ml, 99 mCi/mmol==>5 mCi and 50nmoles.

The toluene is evaporated off until a solution of 10 μl is obtained then25 μl of a solution of DMSO containing 0.2 mg of b-ET-1(19-35) is added,b-ET-1(19-35) (MW=2014.2) having previously been dried over potashovernight. Evaporation is continued for 10 minutes then the solution isagitated under a stream of nitrogen for 15 minutes in order to eliminatethe residual toluene.

Agitation is then gently carried out for 4 days.

To purify, 225 μl of phosphate buffer pH 6.5 and 50 μl of acetonitrileare added then agitation is carried out for 10 minutes and theradioactive solution obtained is separated into 2 injections of 150 μlon a Nucleosil C₁₈ column (150×4.6 mm).

Elution is carried out with a flow rate of 0.8 ml/mn with a gradient of0 to 20% of acetonitrile over 20 minutes then 20 to 35% over 50 minutes.

Analyses:

Analysis of the fractions is carried out by counting the tritium with aliquid scintillation counter (1 μl in 5 ml of scintillating HiSafe3) for60 seconds.

The radioactive fractions are combined and fractionated into 200 μlsamples in siliconized Eppendorf tubes which can be stored at −80° C. or−20° C.

Characteristics of the Product Obtained:

dpm=1 067 274 560 corresponding to 0.5 mCi. Radioactive yield: 10%.Specific activity: (99×10)/30.5=32.5 Ci/mmol.

b)—Determination of the Inhibitory Activity of the Endothelin-convertingEnzyme (ECE)

10 μl of ECE i.e. 1 to 2 μg of purified ECE are pre-incubated for 30minutes at 37° C., in 400 μl of a 50 mM Tris maleate buffer pH=6.5, 20μl of 5M sodium chloride and 5 μl of product P the inhibitory activityof which one wishes to test, in solution at different concentrations(comprised between 1 μM at 100 mM) (i.e. final concentrations in productP of 10 nM to 1 mM).

The reaction is initiated by the addition of 10 μl of(2,3-³H)propionyl-b-ET-1(19-35) prepared as indicated above in a), at afinal concentration of 1.8.10⁻¹²M.

After incubation for one hour at 37° C., the reaction is stopped by theaddition of 600 μl of ethyl acetate and the(2,3-³H)propionyl-b-ET-1(19-21) is extracted by mechanical agitation for2 minutes.

300 μl of the organic phase is removed, 5 ml of liquid scintillator isadded and the radioactivity is counted for 1 minute with a liquidscintillation counter.

Each measurement is carried out in triplicate except for the ECE control(control enzyme, without the product the ECE inhibitory activity ofwhich one wishes to test), for which the measurement is carried out sixtimes.

The percentage inhibition is calculated by establishing therelationship:$\frac{{{tested}\quad {product}} - {blank}}{{{control}\quad {enzyme}} - {blank}}$

The blank is carried out starting with the solution obtained withoutenzyme.

The test was validated by its application to known inhibitors i.e. P1which is phosphoramidon and P2 which isN-(phenylethylphosphonyl)-Leu-Trp (TAKEDA).

The table below gives the results obtained by using as P products theproducts in the examples in the present invention, the ECE inhibitoryactivities of which one wishes to test.

From the cpm's obtained and by plotting the graph of the percentage ofinhibition relative to the concentration of inhibitor (nM), the IC₅₀ iscalculated which therefore corresponds to the concentration which causesa 50% inhibition of ECE.

The numbered results obtained are indicated in the table below:

Products of Examples IC₅₀ (nM) 4 30 6 220 10 150

What is claimed is:
 1. A method of treating vasoconstrictor andhypertension effects in warm-blooded animals comprising administering towarm-blooded animals an amount sufficient to inhibit vasoconstrictor andhypertension effects of a compound of the formula

wherein R₁ is unsubstituted or substituted phenyl or biphenyl, thesubstituents being at least one member of the group consisting ofhydroxy, protected hydroxy, alkoxy of 1 to 4 carbon atoms, —CN, carboxy,salified carboxy, carboxy esterif ied with alkanol of up to 6 carbonatoms, amidified carboxy, benzyloxy and dioxol, n₁ and n₂ areindividually 0 or 1, R₂ is hydrogen or methyl substituted with a memberof the group consisting of phenyl, phenylthio and indolyl, allunsubstituted or substituted by phenyl or substituted with at least onemember of the group consisting of a) halogen, —OH, protected —OH, —CN,alkoxy of 1 to 4 carbon atoms, carboxy, salified carboxy, carboxyesterified with alkanol of 1 to 6 carbon atoms, amidified carboxy,benzyloxy and b) thienyl, naphthyl and phenyl, each unsubstituted orsubstituted with at least one member of the group consisting of halogen,—OH, protected —OH, alkoxy of 1 to 4 carbon atoms, —CN, carboxy,salified carboxy, carboxy esterified with alkanol of 1 to 6 carbon atomsand amidified carboxy, A is selected from the group consisting ofcarboxy, salified carboxy, carboxy esterified with an alkanol of 1 to 6carbon atoms, amidified carboxy, tetrazolyl, salified tetrazolyl andalkyl of 1 to 10 carbon atoms substituted with a member selected fromthe group consisting of a) carboxy, salified carboxy, carboxy esterifiedwith an alkanol of 1 to 6 carbon atoms, amidified carboxy, —OH,protected —OH, alkoxy of 1 to 4 carbon atoms, and b) phenoxy, phenyl,naphthyl, thienyl, indolyl and pyridyl, each unsubstituted orsubstituted with at least one member of the group consisting of halogen,—OH, protected —OH, alkoxy of 1 to 4 carbon atoms, —CN, carboxy,salified carboxy, carboxy esterified with an alkanol of 1 to 6 carbonatoms and amidified carboxy, 1) and 2) indicating possible asymmetriccenters and its addition salts with a non-toxic, pharmaceuticallyacceptable acid or base.
 2. The method of claim 1 wherein n₁ is 1 and itis selected from the group consisting of carboxy, salified carboxy,carboxy esterified with an alkanol of 1 to 6 carbon atoms, amidifiedcarboxy and alkyl of 1 to 10 carbon atoms substituted by a memberselected from the group consisting of carboxy, salified carboxy, carboxyesterified with an alkanol of 1 to 6 carbon atoms, amidified carboxy,—OH, protected —OH, alkoxy of 1 to 4 carbon atoms and phenoxy.
 3. Themethod of claim 1 wherein R₁ is phenyl or biphenyl, both unsubstitutedor substituted with a member selected from the group consisting ofhalogen, —OH and trifluoromethyl sulfonyloxy, n₁ is 1, n₂ is 0, R₂ ishydrogen or methyl substituted with a member of the group consisting ofthienyl, phenyl and cyanophenyl and A is selected from the groupconsisting of carboxy, salified carboxy, carboxy esterified with analkanol of 1 to 6 carbon atoms, amidified carboxy and alkyl of 1 to 10carbon atoms substituted with phenoxy.